Genome Sequencing And Infectious Vector Construction Of Apple Stem Grooving Virus (ASGV)

Apple （Malus domestica Borkh.） is one of the most widely grown fruit crops worldwide.China is the largest producer of apple, both in terms of production and planting area. Shaanxiprovince is the largest apple producer in China, with approximately600000hectares andaccounts for approximately12%of the total production in the world. However, virus diseasessignificantly reduce both yield and quality of apples. The major viruses infecting apple inChina are Apple stem grooving virus （ASGV）, Apple stem pitting virus （ASPV） and Applechlorotic leaf spot virus （ACLSV）. ASGV is the type member of the genus Capillovirus, andis distributed worldwide.In this study at2011to2012, a rapid and sensitive detection method of ASGV wasestablished and the complete genomic sequence of an ASGV isolate from apple wasdetermined, the complete sequence of ASGV is6495bp. Based on the complete or partial （forCP） genomic sequence of ASGV-SHX and of other ASGV isolates, the evolutionaryrelationship and genetic diversity of ASGV isolates were determined. To our knowledge, thisis the first report on the complete genome sequence of ASGV from P. R. China.The study found that ASGV genomic RNA contains two overlapping open readingframes: ORF1（6.3kb） and ORF2（1.0kb）, which encode proteins of241and36kDa,respectively. The large ORF1encodes an apparently of chimeric polyprotein. ORF2encodes aprotein with conserved motifs for both movement proteins （MP） and viral proteases. Thecomplete genome sequence were compared with those of other ASGV isolates from GenBank.ASGV-SHX shared highest nucleotide identity （86%） with the apple isolate from Japan（accession number no. NC₀011749）. The CP gene of ASGV-SHX had the highest identitywith D16681and AB004063（93%with both isolates）. CP gene is the most conserved gene ofASGV.The phylogenetic tree of the complete CP sequences showed that ASGV isolates aredivided into three groups, each containing isolates from China. All the isolates from appleclustered in group1. The isolates in group1were mostly from China, Japan, India, Brazil andKorea. Group2isolates were from China. Group3isolates were mostly from China and theUSA. No clear grouping according to host of isolation was discovered.In order to better understand this virus, we constructed the infection clone of ASGV. Infect chenopodium quina around four weeks by agrobacterium mediated transformationmethod. According to the results of chenopodium quina leaves detection this virus wasdetected in the inoculum leaves, while undetected in the top leaves. It turned out thatinfectious clones constructed could transcribe successfully, but they could not translatesuccessfully. As a result of no cap structure in5’ end of the infectious clones carriers genome,RNA transcribed was degradable and could not be translated successfully. To solve thisproblem we’ll try to construct the complete genome of ASGV to T7Promoter carriers, andthen add a cap analogue in5’ end of the infectious clones carriers genome by using themethod of in vitro transcription, at last verify its activity by infecting chenopodium quina.